Increased BAT Thermogenesis in Male Mouse Apolipoprotein A4 Transgenic Mice

Dietary lipids induce apolipoprotein A4 (APOA4) production and brown adipose tissue (BAT) thermogenesis. Administration of exogenous APOA4 elevates BAT thermogenesis in chow-fed mice, but not high-fat diet (HFD)-fed mice. Chronic feeding of HFD attenuates plasma APOA4 production and BAT thermogenesis in wildtype (WT) mice. In light of these observations, we sought to determine whether steady production of APOA4 could keep BAT thermogenesis elevated, even in the presence of HFD consumption, with an aim toward eventual reduction of body weight, fat mass and plasma lipid levels. Transgenic mice with overexpression of mouse APOA4 in the small intestine (APOA4-Tg mice) produce greater plasma APOA4 than their WT controls, even when fed an atherogenic diet. Thus, we used these mice to investigate the correlation of levels of APOA4 and BAT thermogenesis during HFD consumption. The hypothesis of this study was that overexpression of mouse APOA4 in the small intestine and increased plasma APOA4 production would increase BAT thermogenesis and consequently reduce fat mass and plasma lipids of HFD-fed obese mice. To test this hypothesis, BAT thermogenic proteins, body weight, fat mass, caloric intake, and plasma lipids in male APOA4-Tg mice and WT mice fed either a chow diet or a HFD were measured. When fed a chow diet, APOA4 levels were elevated, plasma triglyceride (TG) levels were reduced, and BAT levels of UCP1 trended upward, while body weight, fat mass, caloric intake, and plasma lipids were comparable between APOA4-Tg and WT mice. After a four-week feeding of HFD, APOA4-Tg mice maintained elevated plasma APOA4 and reduced plasma TG, but UCP1 levels in BAT were significantly elevated in comparison to WT controls; body weight, fat mass and caloric intake were still comparable. After 10-week consumption of HFD, however, while APOA4-Tg mice still exhibited increased plasma APOA4, UCP1 levels and reduced TG levels, a reduction in body weight, fat mass and levels of plasma lipids and leptin were finally observed in comparison to their WT controls and independent of caloric intake. Additionally, APOA4-Tg mice exhibited increased energy expenditure at several time points when measured during the 10-week HFD feeding. Thus, overexpression of APOA4 in the small intestine and maintenance of elevated levels of plasma APOA4 appear to correlate with elevation of UCP1-dependent BAT thermogenesis and subsequent protection against HFD-induced obesity in mice.


Introduction
Obesity has become a global epidemic, affecting more than 40% of adults (nearly 100 million) in the US [1]. It increases the incidence of type 2 diabetes, cardiovascular diseases, stroke, and metabolic disorders [2,3]. It has been found that obese humans and animals have reduced brown adipose tissue (BAT) thermogenesis [4][5][6]. In contrast, activation of BAT thermogenesis reduces body weight, reduces plasma triglycerides (TG), and increases insulin sensitivity in humans and rodents [7][8][9][10][11]. Thus, searching for a potent investigate if maintenance of increased levels of endogenous APOA4 in APOA4-Tg mice could enhance BAT thermogenesis after short-term intake of HFD for 4 weeks or long-term feeding of HFD for 10 weeks, and lead to attenuated body weight gain and plasma lipids and thus protection against HFD-induced obesity. The hypothesis of this study is that overexpression of mouse APOA4 in the small intestine would increase BAT thermogenesis and consequently reduce body weight and plasma lipids in HFD-fed mice. In the present studies, body weight gain, feeding behavior, BAT thermogenic and lipolytic proteins, and plasma parameters in APOA4-Tg and WT mice in response to a standard chow diet or 4-week or 10-week consumption of HFD were investigated.

Body Weight, Fat Mass, Plasma Parameters and BAT Thermogenic and Lipolytic Proteins in APOA4-Tg Mice Fed a Chow Diet
Acute intraperitoneal administration of APOA4 induces sympathetic activity and stimulates BAT thermogenesis in chow-diet-fed mice at normal ambient temperature [31]. To characterize the effect of overexpression of endogenous APOA4 in a control of BAT thermogenesis at normal ambient temperature, body weight and food intake of WT and APOA4-Tg mice fed a chow diet when they were maintained at 21 • C were monitored. After a 5 h fast, plasma APOA4 protein was nearly two-fold higher in APOA4-Tg mice in comparison to WT mice (p < 0.05, Figure 1A). The APOA4-Tg mice produced comparable levels of plasma APOA1 protein relative to WT mice ( Figure 1A). Body weights, caloric intake, total fat and lean mass, and fat mass of interscapular BAT, epididymal white adipose tissue (EWAT), and inguinal white adipose tissue (IWAT) were comparable between the two groups (Table 1). Body weight, caloric intake, and lean/fat mass were monitored when mice (n = 7-8 per group) were maintained on a chow diet at 21 • C. Body weight, tissues, and plasma were collected after a 5 h fast. Daily caloric intake is the average of seven days of caloric intake measured during the 4th week of chow diet feeding. Values are represented as mean ± SEM. Values with asterisks (*) represent significant differences relative to the WT mice (p < 0.05).
To examine whether the induction of norepinephrine synthesis by overexpression of APOA4 leads to activated AMPK pathway and enhanced intracellular lipolysis and BAT thermogenesis, levels of UCP1, TH, and AMPK pathway and lipolytic enzymes in BAT were determined. Protein levels of UCP1, TH, ATGL, and HSL in BAT were not significantly different between APOA4-Tg mice and their WT controls when maintained on a chow diet at 21 • C ( Figure 1B-E). Similarly, no significant differences in levels of Ucp1, Ucp3, Atgl, Hsl, and Ampkα1 mRNA in the BAT were observed between APOA4-Tg and WT mice ( Figure 1F). Ucp3, Atgl, Hsl, and Ampkα1 mRNA in the BAT were observed between APOA4-Tg and WT mice ( Figure 1F). Plasma and BAT in 5 h fasted mice were collected. Plasma apolipoproteins in WT mice were considered as 100%. Data are expressed as mean ± SEM for 6-8 animals per group. Values with asterisks (*) represent significant differences relative to the WT mice (p < 0.05). To test if overexpression of APOA4 alters energy expenditure and locomotor activity, energy expenditure, respiratory exchange ratio (RER), and locomotor activity in chow-fed APOA4-Tg mice were measured. When fed a chow diet, APOA4-Tg mice had comparable lean body mass (22.8 ± 1.3 g) not normalized by body weight relative to WT mice (21.5 ± 0.9 g). No significant differences in hourly EE, average of EE in light or dark phases or overall, RER, and locomotor activity were observed between APOA4-Tg and WT mice when fed a chow diet (Figure 2A-D). APOA4-Tg mice did have reduced levels of plasma TG compared to WT mice (p < 0.05; Table 1); levels of plasma cholesterol and leptin were comparable between APOA4-Tg and WT mice (Table 1). Thus, APOA4-Tg mice have elevated plasma APOA4, reduced plasma TG, and comparable levels of UCP1-dependent BAT thermogenesis (as indicated by BAT UCP1 levels), energy expenditure, body weight, caloric intake, fat mass, plasma cholesterol, and plasma leptin when they are maintained on a chow diet at 21 • C.
To test if overexpression of APOA4 alters energy expenditure and locomotor a energy expenditure, respiratory exchange ratio (RER), and locomotor activity in ch APOA4-Tg mice were measured. When fed a chow diet, APOA4-Tg mice had comp lean body mass (22.8 ± 1.3 g) not normalized by body weight relative to WT mice 0.9 g). No significant differences in hourly EE, average of EE in light or dark ph overall, RER, and locomotor activity were observed between APOA4-Tg and W when fed a chow diet (Figure 2A-D). APOA4-Tg mice did have reduced levels of p TG compared to WT mice (p < 0.05; Table 1); levels of plasma cholesterol and lepti comparable between APOA4-Tg and WT mice (Table 1). Thus, APOA4-Tg mice ha vated plasma APOA4, reduced plasma TG, and comparable levels of UCP1-dep BAT thermogenesis (as indicated by BAT UCP1 levels), energy expenditure, body w caloric intake, fat mass, plasma cholesterol, and plasma leptin when they are main on a chow diet at 21 °C.

Body Weight, Fat Mass, Plasma Parameters, and BAT Thermogenic and Lipolytic Proteins in APOA4-Tg Mice Fed an HFD for 4 Weeks
To investigate whether increased levels of endogenous APOA4 can modulate BAT thermogenesis and body weight after short-term consumption of an HFD, body weight, fat mass, caloric intake, BAT thermogenic and lipolytic proteins, and plasma lipids in WT and APOA4-Tg mice were measured when they were fed an HFD for 4 weeks. To minimize temperature-induced BAT thermogenesis, APOA4-Tg and WT mice were maintained at a thermoneutral housing temperature (28 ± 0.5 • C, minimal BAT activity) [15]. Our preliminary data showed that this increased temperature had no effect on body weight, food intake, and plasma lipids in APOA4-Tg mice in comparison with WT mice when mice were fed a chow diet at 28 • C. After 4 weeks of HFD, APOA4-Tg mice had higher levels of plasma APOA4 than WT mice (p < 0.05; Figure 3A). In contrast, plasma APOA1 protein levels were similar between the two genotypes ( Figure 3A). APOA4-Tg mice had significantly higher levels of BAT UCP1 than WT mice (p < 0.05; Figure 3B). No significant differences in protein levels of TH, ATGL, and HSL in BAT, and BAT mRNA of Ucp1, Ucp3, Atgl, Hsl, and Ampkα1 were observed between APOA4-Tg and WT mice after 4 weeks of an HFD ( Figure 3C-E). During the 4 weeks of HFD feeding, the average weekly body weights of the APOA4-Tg and WT mice were comparable ( Figure 3G). After 4 weeks of HFD feeding, APOA4-Tg mice exhibited comparable body weights, average daily caloric intake, total fat and lean mass, and fat mass of interscapular BAT, EWAT, and IWAT relative to the WT mice ( Table 2). In addition, APOA4-Tg mice had reduced levels of plasma TG (p < 0.05, Table 2) and comparable levels of plasma cholesterol and leptin in response to 4 weeks of HFD ( Table 2). The findings suggest that plasma APOA4 levels in APOA4-Tg mice over-expressing APOA4 are not suppressed by short-term feeding of an HFD, and they elevate UCP1-dependent BAT thermogenesis and reduce plasma TG as compared to WT mice when housed under thermoneutral conditions, but do not alter body weight, caloric intake, and levels of plasma cholesterol and leptin. To investigate whether endogenous APOA4 production in APOA4-Tg mice can be induced by chronic consumption of an HFD, and whether increased levels of endogenous APOA4 can modulate BAT thermogenesis and lipid metabolism, BAT thermogenic and lipolytic proteins, body weight, caloric intake, and plasma lipids in WT and APOA4-Tg mice were measured when they were fed an HFD for 10 weeks at 28 • C. APOA4-Tg mice produced nearly three-fold more plasma APOA4 and two-fold more APOA1 compared with WT mice (p < 0.05; Figure 4A). Levels of UCP1, TH, and ATGL proteins in the BAT of APOA4-Tg mice were significantly greater than those in WT mice (p < 0.05, Figure 4B-D). APOA4-Tg mice had a tendency toward increased BAT HSL protein levels relative to WT mice ( Figure 4E). Relative to WT mice, APOA4-Tg mice had upregulated levels of Ucp1, Atgl, and Ampkα1 gene expression in BAT (p< 0.05, Figure 4F). No significant differences in Ucp3 and Hsl gene expression levels in BAT were observed between APOA4-Tg and WT mice ( Figure 4F).
Body weights of WT and APOA4-Tg mice increased throughout the 10-week feeding ( Figure 5A). During the first 3 weeks of the HFD, weekly body weights of APOA4-Tg mice were comparable to WT mice ( Figure 5A). In contrast, body weights of APOA4-Tg mice were significantly less than WT mice in the last 7 weeks of feeding (p < 0.05; Figure 5A). By the end of the 10-week feeding of the HFD, significant reductions in body weight gain and fat mass, especially fat mass of BAT and EWAT, were observed in the APOA4-Tg mice in comparison with WT mice (p < 0.05; Figure 5B,C and Table 3). levels of plasma TG (p < 0.05, Table 2) and comparable levels of plasma choles leptin in response to 4 weeks of HFD ( Table 2). The findings suggest that plasm levels in APOA4-Tg mice over-expressing APOA4 are not suppressed by short-t ing of an HFD, and they elevate UCP1-dependent BAT thermogenesis and redu TG as compared to WT mice when housed under thermoneutral conditions, b alter body weight, caloric intake, and levels of plasma cholesterol and leptin. G. Weekly body weight-4-week HFD   Body weight, caloric intake, and lean/fat mass were monitored when mice (n = 6-7 per group) were fed an HFD for 4 weeks at 28 • C. Body weight, tissues, and plasma were collected after a 5 h fast. Daily caloric intake is the average of seven days of caloric intake measured during the 4th week of HFD feeding. Values are represented as mean ± SEM. Values with asterisks (*) represent significant differences relative to the WT mice (p < 0.05).
To examine if overexpression of APOA4 regulates energy expenditure in HFD-fed mice, energy expenditure, RER, and locomotor activity of WT and APOA4-Tg mice fed 10 weeks of an HFD were determined. In contrast to the percent lean mass normalized by body weight (Figure 5C), no significant difference in lean body mass not normalized by body weight between APOA4-Tg mice (21.3 ± 1.0 g) and WT mice (20.3 ± 0.8 g) was observed. Relative to the WT mice, APOA4-Tg mice fed an HFD exhibited increased energy expenditure at several time points during HFD feeding (p < 0.05, Figure 6A), but no significant difference in average of EE in light or dark phases or overall were observed between WT and APOA4-Tg mice ( Figure 6B). Additionally, RER and locomotor activity in these APOA4-Tg mice were comparable to their controls ( Figure 6C,D). In addition, plasma levels of TG and leptin in APOA4-Tg mice were lower than in WT mice (p < 0.05; Table 3). Daily caloric intake and plasma cholesterol levels were similar between the APOA4-Tg and WT mice (Table 3). These findings suggest that chronic consumption of an HFD induces plasma APOA4 levels in APOA4-Tg mice and the action of APOA4 stimulates norepinephrine levels and AMPKα-dependent pathway, elevates intracellular lipolysis, UCP1-dependent BAT thermogenesis and energy expenditure at several time points, attenuates an HFD-induced rise in body weight gain, fat mass and plasma lipids, and are independent of caloric intake after 10 weeks of HFD feeding.
lipolytic proteins, body weight, caloric intake, and plasma lipids in WT and APOA4-Tg mice were measured when they were fed an HFD for 10 weeks at 28 °C. APOA4-Tg mice produced nearly three-fold more plasma APOA4 and two-fold more APOA1 compared with WT mice (p < 0.05; Figure 4A). Levels of UCP1, TH, and ATGL proteins in the BAT of APOA4-Tg mice were significantly greater than those in WT mice (p < 0.05, Figure 4B-D). APOA4-Tg mice had a tendency toward increased BAT HSL protein levels relative to WT mice ( Figure 4E). Relative to WT mice, APOA4-Tg mice had upregulated levels of Ucp1, Atgl, and Ampkα1 gene expression in BAT (p< 0.05, Figure 4F). No significant differences in Ucp3 and Hsl gene expression levels in BAT were observed between APOA4-Tg and WT mice ( Figure 4F).  Body weights of WT and APOA4-Tg mice increased throughout the 10-week feeding ( Figure 5A). During the first 3 weeks of the HFD, weekly body weights of APOA4-Tg mice were comparable to WT mice ( Figure 5A). In contrast, body weights of APOA4-Tg mice were significantly less than WT mice in the last 7 weeks of feeding (p < 0.05; Figure 5A).   Body weight and caloric intake were monitored when mice (n = 9-10 per group) received an for 10 weeks at 28 °C. Body weight, tissues, and plasma were collected after a 5 h fast. Daily c  Body weight and caloric intake were monitored when mice (n = 9-10 per group) received an HFD for 10 weeks at 28 • C. Body weight, tissues, and plasma were collected after a 5 h fast. Daily caloric intake is the average of seven days of caloric intake measured during the 10th week of HFD feeding. Values are represented as mean ± SEM. Values with asterisks (*) represent significant differences relative to the WT mice (p < 0.05).
ulates norepinephrine levels and AMPKα-dependent pathway, elevates intrac polysis, UCP1-dependent BAT thermogenesis and energy expenditure at sev points, attenuates an HFD-induced rise in body weight gain, fat mass and plasm and are independent of caloric intake after 10 weeks of HFD feeding.

Discussion
Although APOA4 is a well-known satiating protein that enhances thermoge fatty acid uptake in BAT of chow-fed mice [31,38], the role of APOA4 in the regu BAT thermogenesis and lipid metabolism in HFD-fed obese mice remained e part because APOA4 levels are normally suppressed by chronic feeding of an H 36]. The present experiments tested the hypothesis that non-suppressible expr

Discussion
Although APOA4 is a well-known satiating protein that enhances thermogenesis and fatty acid uptake in BAT of chow-fed mice [31,38], the role of APOA4 in the regulation of BAT thermogenesis and lipid metabolism in HFD-fed obese mice remained elusive, in part because APOA4 levels are normally suppressed by chronic feeding of an HFD [34][35][36]. The present experiments tested the hypothesis that non-suppressible expression of mouse APOA4 in the small intestine, with a subsequent rise in plasma APOA4, would increase BAT thermogenesis and consequently reduce body weight and plasma lipids in HFD-fed mice. This series of experiments demonstrated that male APOA4-Tg mice maintained increased levels of plasma APOA4, correlating with increased UCP1-dependent BAT thermogenesis (reflected as an increase in BAT UCP1), in particular when fed an HFD, as well as a decrease in plasma TGs, which over the course of 10 weeks also resulted in reduced plasma lipid levels, body weight, and fat mass in comparison with their WT control group on the same diet, all without altering caloric intake. Since BAT function is sex-dependent [39], the effect of APOA4 in female APOA4-Tg mice remains to be tested.
In response to dietary challenge, UCP1 expression in BAT is increased in mice [40]. HFD induces BAT UCP1 levels [40]. Our previous reports demonstrated that APOA4-KO mice exhibited reduced levels of UCP1-dependent BAT thermogenesis in response to acute feeding of dietary lipids or one week of HFD feeding [32,41], demonstrating that peripheral and/or central APOA4 may elevate UCP1-dependent BAT thermogenesis. In the current experiments, APOA4-Tg mice had comparable UCP1-dependent BAT thermogenesis, energy expenditure, and food intake relative to their WT controls when they were maintained on chow diets at 21 • C. When fed an HFD for 10 weeks and housed at 21 • C, no significant differences in weekly body weight, body weight gain, and daily food intake were observed between APOA4-Tg and WT mice (Supplementary Figure S1). Mice have minimal BAT activity at thermoneutrality, the range of ambient temperatures without regulatory changes in metabolic heat production or heat loss [15]. Because the BAT activity of chowor HFD-fed mice housed at 21 • C was activated to produce extra heat for defending their body temperature, the possibility existed that the lack of difference in body weight gain between APOA4-Tg and WT mice fed 10 weeks of HFD was due to extra heat production for defending their body temperature when housed at 21 • C. Thus, APOA4-Tg and WT mice were housed at thermoneutrality (28-30 • C) for minimized induction of BAT activity by ambient temperature in the HFD feeding experiments. Chow-fed APOA4-Tg mice had comparable body weight, fat mass, and caloric intake relative to their WT mice when they were housed at 28 • C (our unpublished data).
APOA4 KO mice have impaired lipid-induced norepinephrine synthesis and UCP1dependent BAT thermogenesis [32]. In contrast, acute injection of APOA4 stimulates sympathetic activity, norepinephrine synthesis, intracellular lipolysis, and BAT thermogenesis in BAT of chow-fed mice in response to dietary lipids [31]. The findings suggest that APOA4 may induce UCP1-dependent BAT thermogenesis through elevation of norepinephrine levels and intracellular lipolysis. In the current study, overexpression of APOA4 elevated UCP1-dependent BAT thermogenesis, as indicated by increased levels of BAT UCP1, but did not alter norepinephrine synthesis (as indicated by levels of TH), AMPKα pathway, intracellular lipolysis, body weight, and fat mass in mice after short-term intake of an HFD for 4 weeks. Although whether overexpression of APOA4 in the small intestine would result in the stimulation of sympathetic activity in BAT in HFD-fed mice remains unknown, the present study indicated that after chronic consumption of an HFD for 10 weeks, APOA4-Tg mice had increased norepinephrine synthesis, ATGL-induced intracellular lipolysis, and elevated UCP1-dependent BAT thermogenesis in HFD-fed mice. The findings suggest that overexpression of APOA4 may act on sympathetic nerves, leading to increased UCP1-dependent BAT thermogenesis through activation of an AMPKα1-dependent pathway for increased ATGL-dependent intracellular lipolysis.
APOA4-KO mice had attenuated energy expenditure and comparable locomotor activity after one-week or 20-week feeding of HFD at 21 • C [32,41], indicating that APOA4 may enhance energy expenditure but does not alter locomotor activity. The current experiment demonstrated that APOA4-Tg exhibited an increase in energy expenditure at several time points, but no alteration in the average of overall energy expenditure independent of locomotor activity when fed 10 weeks of an HFD at 30 • C. Although overexpression of APOA4 increased plasma APOA4 levels in the current experiment, and a central effect of APOA4 has been shown to elevate BAT thermogenesis [42], it is unlikely in the current experiments that APOA4-Tg mice exhibited enhanced BAT thermogenesis and energy expenditure induced by central APOA4, because APOA4 produced in the intestine and found in plasma of APOA4-Tg mice cannot cross the blood brain barrier [43]. Consistent with previous findings [27,32,38], the present study indicated that overexpression of APOA4 did not inhibit daily caloric intake in APOA4-Tg mice when they were maintained on a chow diet, 4 weeks of an HFD, or 10 weeks of an HFD. APOA4-KO mice had comparable RER in our previous report [32]. The current study demonstrated that APOA4-Tg mice had comparable RER when fed a chow diet or an HFD, suggesting that no significant differences in energy substrates are used for heat production. During 10 weeks of HFD feeding, APOA4-Tg mice exhibited reduced body weight starting at 4 weeks. In contrast, APOA4-Tg mice had comparable body weight to their WT controls during 4 weeks of HFD feeding. This difference may have been due to the difference in the ages of the mice at the start of the HFD feeding. Mice were 10 weeks of age at the start of HFD feeding in the 10-week study while mice were 16 weeks of age at the start of HFD feeding in the 4-week study. The findings suggest that the reduction of body weight gain in APOA4-Tg mice with elevation of energy expenditure at several time points is independent of caloric intake and locomotor activity after 10 weeks of HFD.
Enhancement of BAT thermogenesis reduces hypertriglyceridemia and protects against obesity-related atherosclerosis development [9,[44][45][46][47]. TG in chylomicrons or very lowdensity lipoprotein (VLDL) are hydrolyzed by action of lipoprotein lipase (LPL), and free fatty acids are released to the circulation [48]. Sympathetic activation leads to upregulation of LPL activity [49,50]. APOA4 has been reported to activate BAT sympathetic activity and promote LPL-induced hydrolysis of triglyceride-rich lipoproteins [30,31,51]. BAT is the major fat depot for APOA4-induced fatty acid uptake through elevation of LPL expression [31]. APOA4-KO mice have normal fat absorption, VLDL production, and comparable levels of plasma triglyceride and leptin, but delayed chylomicron clearance, impaired proximal TG transport, and altered intestinal gene expressions related to TG transport when compared with their WT control group [32,41,[52][53][54]. However, whether alterations in fat absorption, chylomicron clearance, TG transport, and intestinal gene expression would also be observed in APOA4-Tg mice remains unknown. The effect of overexpression of intestinal APOA4 in the attenuation of HFD-induced obesity through modulation of lipid transport and metabolism in the small intestine, adipose tissues, and liver needs to be investigated.
APOA4 overexpression did not significantly affect fasting levels of triglyceride and cholesterol in chow-fed mice at normal ambient temperature in a previous study [29]. In the current study, overexpression of endogenous APOA4 did not alter plasma cholesterol and leptin levels, though a reduction in plasma TG was observed when the APOA4-Tg mice were maintained on a chow diet at normal temperature. The difference in plasma TG levels in chow-fed APOA4-Tg mice between the current and previous study could be due to differences in genetic background resulting from more extensive C57BL/6J backcrossing, various environmental factors, or dietary lipid composition of the chow diets [29]. After feeding of atherogenic diet for 5 days, comparable plasma TG and cholesterol were found between these two genotypes [29]. When fed an atherogenic diet for 14 weeks, APOA4-Tg mice exhibited increased levels of plasma TG and cholesterol compared to WT males [29]. Thus, chronic feeding of the atherogenic diet may affect plasma lipids in APOA4-Tg mice. In the present experiment, after short-term intake of HFD for 4 weeks, overexpression of APOA4 attenuated plasma TG, possibly due to APOA4-induced fatty acid uptake by BAT [31]. Furthermore, plasma leptin and cholesterol in the APOA4-Tg mice were not altered after short-term intake of HFD. The current experiment demonstrated that chronic consumption of HFD increased plasma lipids and leptin in WT mice, consistent with the observations in previous reports [55,56]. In contrast, APOA4-Tg mice exhibited attenuation of an HFD-induced rise in the plasma lipids and leptin. It is possible that APOA4-Tg mice have reduced plasma TG resulting from elevated LPL activity in BAT which promote triglyceride clearance and fatty acid uptake by BAT. A more extensive, comparative analysis of chow-and HFD-fed mice at room temperature and thermoneutrality is needed to provide better insight into the lipid profiles of APOA4-Tg mice, while investigation of the effect of endogenous APOA4 in the downregulation of plasma lipids through upregulation of fatty acid uptake and LPL activity in BAT of APOA4-Tg mice is also required.
A previous report has shown that APOA1 also activates BAT thermogenesis and consequently reduces body weight gain and fat mass in HFD-induced obese mice [57]. In the present study, because chronic consumption of HFD for 10 weeks increased levels of plasma APOA4 and also APOA1 in APOA4-Tg mice, APOA4-Tg mice likely experience a mixed effect of APOA4 and APOA1 in elevation of BAT thermogenesis and downregulation of body weight gain and fat mass after 10 weeks of HFD feeding. When chylomicrons enter the circulation, approximately 25% of the APOA4 is transferred to high-density lipoprotein (HDL) [58]. APOA1 is the most abundant apolipoprotein in an HDL [59]. APOA4 and APOA1 promote reverse cholesterol transport from extrahepatic cells and tissues to the liver and intestine for excretion [25,58,59].In the APOA4-Tg mice after 10 weeks of HFD feeding, overexpression of APOA4 elevated levels of plasma APOA4 and APOA1 and consequently downregulated levels of plasma cholesterol, possibly due to the mixed effect of APOA4 and APOA1 in facilitating reverse cholesterol transport. Further investigation into the elevation of reverse cholesterol transport in APOA4-Tg mice is required.
Activating BAT thermogenesis in rodents and humans has a great potential to combat obesity and cardiovascular diseases [7,9,10]. In view of the observations made using APOA4-KO mice previously [32] and using APOA4-Tg mice in the current study, lipidinduced endogenous APOA4 may increase UCP1-dependent BAT thermogenesis and energy expenditure at several time points and reduce body weight gain, fat mass and plasma lipids. Collectively, the present findings suggest that increased levels of endogenous APOA4 can elevate UCP1-dependent BAT thermogenesis and consequently attenuate HFDinduced rises in body weight gain, fat mass and plasma lipids in obese mice independent of caloric intake.

Animals
Male APOA4-Tg mice and WT mice (C57BL/6J background) were generated in an AAALAC-accredited facility under conditions of controlled illumination (12:12 h light-dark cycle, lights on from 0600 to 1800 h). APOA4-Tg mice were kindly provided by Dr. Karen Reue at the University of California, Los Angeles, CA, USA [29] and were back-crossed for >10 generations onto a C57BL/6J genetic background. All mice were genotyped by polymerase chain reaction (PCR) analysis of tail deoxyribonucleic acid (DNA) [29]. All animals at ages between 10 and 20 weeks received free access to water and either a standard chow diet (14% fat by weight, 60% carbohydrates by weight, and 25% protein by weight; # Prolab RMH3000 5P00, LabDiet, St. Louis, MO, USA) or an HFD (60% lard fat by weight, 20% carbohydrates by weight, and 20% protein by weight; # D12492, Research Diets, Inc., New Brunswick, NJ, USA) for 4 or 10 weeks. Body weight was monitored with a toploading balance (± 0.01 g, Adenturer SL, Ohaus Corp, Pine Brook, NJ, USA). All animal protocols were approved by the Institutional Animal Care and Use Committee at Ohio University and University of Cincinnati.

Body Weight, Fat Mass, Plasma Parameters, and BAT Thermogenic and Lipolytic Proteins in APOA4-Tg Mice Fed a Chow Diet
WT and APOA4-Tg mice (n = 7-8 per group) at the age of 15 weeks had free access to a standard chow diet and water for 4 weeks while housed at 21 • C. Body weight and caloric intake were recorded weekly. At the end of the experiment, body composition was measured by LF-50 body composition analyzer (Bruker, Billerica, MA, USA) and body weight was recorded. Food was removed for 5 h before plasma, interscapular BAT, EWAT, and IWAT were collected and stored at −80 • C for further determinations.

Body Weight, Fat Mass, Plasma Parameters and BAT Thermogenic and Lipolytic Proteins in APOA4-Tg Mice Fed an HFD for 4 Weeks
Body weight and caloric intake in WT and APOA4-Tg mice (n = 6-8 per group) at the age of 16 weeks were recorded weekly while maintained on an HFD for 4 weeks at 28 • C. At the end of the experiment, body composition was measured by LF-50 body composition analyzer. Food was removed for 5 h before plasma, interscapular BAT, EWAT, and IWAT were collected and stored at −80 • C for further determinations.

Body Weight, Fat Mass, Plasma Parameters, and BAT Thermogenic and Lipolytic Proteins in APOA4-Tg Mice Fed an HFD for 10 Weeks
WT and APOA4-Tg mice (n = 9-10 per group) at the age of 10 weeks received 10 weeks of HFD feeding while housed at 28 • C. At the end of the experiment, body composition was measured by LF-50 body composition analyzer. Food was removed for 5 h before plasma, interscapular BAT, EWAT, and IWAT were collected and stored at −80 • C for further determinations.

Plasma Parameters
Plasma leptin level was determined using a commercial mouse leptin ELISA kit (Crystal Chem, Elk Grove Village, IL, USA). Briefly, plasma (10 µL) was added to each well of a microtiter plate pre-coated with anti-leptin monoclonal antibodies, and the detection antibody was added. After incubation, absorbance was measured with a microplate reader (Synergy HT, BioTek Instruments, Inc., Richmond, VA, USA), and the final concentrations were calculated using standards provided with the ELISA kit. Triglyceride and cholesterol in the plasma were determined using Infinity triglyceride and cholesterol kits (Thermo Scientific, Middletown, VA, USA). 4.7. Energy Expenditure, RER, and Food Intake of Mice Fed a Chow or HFD for 10 Weeks Energy expenditure, RER, locomotor activity, and food intake were determined in APOA4-Tg and WT mice at an age of 19 weeks fed a powdered chow (n = 8 per group), or during the 10th week of HFD feeding (n = 4 per group). These animals were acclima-tized to individual metabolic cages of a comprehensive lab animal monitoring system (CLAMS, Columbus Instrument, Columbus, OH, USA) for 3 days. Energy expenditure, RER, locomotor activity, and food intake were then recorded at 16 min intervals for 2 days.

Quantitative RT-PCR
Total RNA from BAT of 5 h fasted mice was isolated using a RNeasy lipid tissue mini kit (Qiagen, Hidden, Germany) according to the manufacture's protocol, and firststrand complementary DNA (cDNA) was synthesized from 1 µg total RNA using a iScript cDNA synthesis kit (Bio-Rad Laboratories) [60]. The sequences of the primers (Integrated DNA Technologies, Coralville, IA, USA) were according to our published protocols [32,42]. Quantitative PCR (qPCR) was performed in a 25 µL final reaction volume, including 4µL of 10-fold diluted sample cDNA, 1µL of 10µM forwarded or reverse primers, and iTaq universal SYBR green supermix (Bio-Rad Laboratories), using a Bio-Rad RT-PCR instrument. PCR conditions were conducted as follows: 95 • C for 10 min for one cycle, followed by 40 cycles of 95 • C for 15 s and 60 • C for 60 s. Threshold cycle readings for each of the unknown samples were obtained, and the results were analyzed in Excel using the ∆∆Ct method [35]. Levels of 36B4 mRNA from each sample were similar among all groups and were used as internal controls to normalize the mRNA levels.

Statistical Analysis
All values are presented as mean ± SEM. For body weight measurements, repeated measures analyses of variance (ANOVA), followed by a Sidak's multiple comparisons test, were performed using GraphPad™ Prism (version 9.0, San Diego, CA, USA). For energy expenditure measurements, analysis of covariance (ANCOVA) was performed with lean mass as the covariate using SPSS version 28.0 software (SPSS Inc., Chicago, IL, USA) [61,62]. For end-point measurements, unpaired t-tests were performed using GraphPad™ Prism (version 9.0, San Diego, CA, USA). Differences were considered significant if the p value was <0.05.

Conclusions
Maintenance of mouse APOA4 levels in the small intestine and plasma elevates UCP1dependent BAT thermogenesis and energy expenditure and attenuates HFD-induced gains in body weight, fat mass, and plasma lipids, leading to protection against HFD-induced obesity in mice.